Apoptosis is a programmed physiological mode of cell death that eliminates compromised or superfluous cells and that plays an important role in tissue homeostasis.
Apoptosis is involved in pathological conditions in which the delicate balance between cell proliferation and death is disturbed. Apoptosis can be induced by endocrine and other stimuli, negative selection in the immune system and a substantial proportion of T-cell killing. It also accounts for many cell deaths following exposure to cytotoxic compounds, hypoxia or viral infection. It is a major factor in the cell kinetics of tumours, both growing and regressing.
Many anti-cancer agents exert their effects through initiation of apoptosis, and even the process of carcinogenesis itself may depend upon a selective critical failure of apoptosis that permits the survival of cells after critical mutagenic DNA damage.
Apoptosis probably contributes to many chronic degenerative processes including Alzheimer's disease, Parkinson's disease and heart failure. Because of efficient multifactorial mechanism of cell death, apoptosis itself does not induce inflammatory response in vivo.
In contrast to apoptosis, necrosis is an accidental cell death due to chemical or physical injury of the cell membrane. Morphological criteria of necrosis include cell swelling (instead of shrinking), cell lysis, lysosomal leakage and loss of the membrane integrity. The cellular changes that are characteristic for apoptosis may also be found for necrosis.
Necrosis in pathology occurs when cells are exposed to extreme variance from physiological conditions (e.g.; hypothermia, hypoxia, strong UV and ionising radiation), which result in damage of plasma membrane. Necrosis is commonly accompanied by intense inflammatory response and tissue damage because of the leakage of the lysosomal enzymes into extra cellular fluid.
It is known that normal cells exhibit remarkable asymmetry of lipid distribution between inner and outer leaflets of cell membranes, which is lost during the early steps of apoptosis and necrosis. Most characteristic in this change is the exposure to cell surface of amine-containing phospholipids such as phosphatidylethanolamine (PE) and phosphatidylserine (PS). This exposure is functionally important as it provides the signal for recognition and elimination of apoptotic cells by macrophages. This change of cell membrane properties allows also to identify and characterize apoptotic cells.
One of the first approaches for the determination of cell apoptosis was based on the detection of the exposure of amino groups on the cell surface that can be detected by chemical reagents specific for amino groups. However, the low specificity of these reactions and their strong dependence on the environment limit their application in apoptosis research.
Therefore, more specific methods based on molecular recognition of surface-exposed PS and PE were developed.
The most popular of them is based on the property of annexin V to interact with PS exposed on the surface in a Ca2+-dependent manner. Different variants of this method were developed. For instance, in one of this variant, annexin V labelled with fluorescein was used for flow cytometry while annexin V labeled with red-near infrared dyes is used for tissue imaging. This protein was also labeled with colloid gold for electron microscopy, with radioactive tracer for autoradiography on the tissue level and with peroxidase for histochemical studies. In all these tests, a high (up to 2.5 mM) extracellular concentration of Ca2+ ions has to be provided for complete binding of annexin V to PS. Since Ca2+ ions activate the protein scramblase that randomises the phospholipids distribution, this enzyme can move PS to the cell surface in a calcium-dependent manner and lead to false positive results.
Furthermore, annexin V can associate with membrane surfaces containing by-products of lipid per oxidation that modify amines by producing negative charges.
Moreover, detergents in the medium can also change the annexin V lipid binding specificity.
In addition, routinely used cell harvesting techniques for adhering cells, such as trypsinization, can also produce false results in application of this method.
Finally, for complete annexin V binding, pre-incubation times of up to 1 h are in principle needed, making kinetic measurements problematic.
Alternative methods include application of monoclonal antibodies against negatively charged lipids. However, these antibodies found a limited application, probably because of their lability, high cost and complicated procedures for visualization of their binding. Moreover, it was shown that their binding inhibits Na/K-ATPase activity. Meantime, mimicking the behaviour of antibodies allowed to find peptides that can specifically recognize and bind PS and PE. Biotinilated peptides can be conjugated with fluorescently labeled streptavidin providing fluorescent labeling of apoptotic cells.
Tracing of lipid exchange between leaflets can also be made with the aid of fluorescently labeled lipids. However, intervening in the lipid distribution between leaflets is not well tolerated by the cells, and cell treatment with exogenic PS can itself induce apoptosis.
Probes for the exposure of PS on cell surface can be designed by synthetic organic chemistry. For instance, an organic molecule composed of a fluorophore and an artificial zinc-containing receptor for PS recognition was suggested for apoptosis detection. Providing a significant simplification of the detection procedure, this probe is still far from the ideal solution for detecting apoptotic cells, mainly because of the requirement for the presence of chelating ions in the medium.
The increase of the negative charge of apoptotic cells due to PS exposure can also be used for sensing. Cationic liposomes with incorporated fluorescent phospholipid analogs bind to apoptotic cells and provide their labeling. Based on the same principle of selective binding to negatively charged surface, some positively charged nanoparticles have been selected from chemically derived library.
The membrane-specific detection of apoptotic cells can be based on a quite different concept. The randomisation of lipid content should provide a decrease of lipid order and membrane rigidity. The small size of the lipid head of PE and the repulsion between negatively charged PS heads should increase hydration of the outer leaflet, and these changes can be detected by fluorescent dyes.
There are many observations that apoptotic cells exhibit increased binding of not only cationic amphiphilic drugs, such as chlorpromazine and verapamil, but also of the negatively charged lipophilic dye, merocyanine 540 (M540). This dye is known to bind most efficiently to structurally destabilized lipid bilayers and therefore can distinguish apoptotic cells. However, the approach based on M540 binding is rarely used because of its low specificity.
Therefore, another approach for the detection of cell apoptosis based on the change of order/hydration in the outer leaflet of cell membrane has been developed. This approach is focused on the unique properties of 3-hydroxyflavone (3HF) derivatives as environment-sensing dyes with two-color ratiometric response. These dyes were functionalised for binding to phospholipids membranes. This allowed observing a strong two-color response of fluorescence emission to variation of phospholipids composition. The most significant effect was due to variation of the surface charge observed in comparison between neutral and negatively charged phospholipids bilayers.
However, these 3-hydroxyflavone (3HF) derivatives are characterized by a rapid penetration inside the cells and present a low selectivity to cell plasma membranes.
The present inventors have thus underlined that the key problem for the selectivity of these 3-hydroxyflavone (3HF) compounds was to incorporate the dye selectively into the outer leaflet of the plasma membrane. The inventors' work demonstrated that this aim could be realized by coupling a long hydrocarbon chain and a zwitterionic group to the 3-HF moiety, in order to mimic the lipid structure and to place the fluorophore close to the membrane surface. The inventors have thus developed a new 3-HF compound that possesses a zwitterionic group and a long hydrocarbon chain which significantly diminishes the penetration rates of the probes through a bilayer. As a consequence, this compound is not redistributed into the cell interior and remains located in the cell plasma membrane.
Experiments by the present inventors have shown that, compared with the known non-invasive methods for the detection of apoptosis, the use of the new 3-HF compound as a probe for the detection of cell apoptosis presents the following advantages:                it is easier to handle and to prepare,        it enables quantitative measurements that are independent on the local probe concentration,        it requires shorter incubation times, and        its response to apoptosis does not depend on the presence of Ca2+ ions or on any other compound present in the cell environment.        
The present invention will become better understood and other aspects, advantages, objectives of the present invention will become apparent from the following description taken in close conjunction with the accompanying figures. These are for illustration only, and thus are not to be considered as limiting the present invention.